Asymmetry in oocytes is a well-documented and conserved feature among animals including vertebrates and humans. In vertebrates, the earliest indicator of cell polarity is an asymmetric aggregate, known as the Balbiani body, that includes organelles, proteins, and, in some animals, mRNAs encoding germline determinants. In non-mammalian vertebrates, this early asymmetry is known to indicate the animal-vegetal axis, but the relationship between the Balbiani body and the animal-vegetal axis in mammals is not understood. Although the animal- vegetal axis is the first axis to form in vertebrates, and is crucial for normal development of the embryonic axes that form later in development, its specification is poorly understood. In a maternal-effect genetic screen we isolated 2 alleles of bucky ball (buc), mutants that lacks oocyte asymmetry and fails to establish the axes in embryos. The Buc protein does not contain any characterized or known functional domains based on sequence comparison, but other vertebrates including humans have bucky ball genes. The buc gene, and our mutant alleles provide the first genetic access and a unique entry point to the developmental pathway regulating oocyte polarity. Here three aims are proposed to study how cell polarity is established and maintained in the vertebrate ovary. 1) We will test the hypothesis that buc specifies the oocyte axis upstream or at the level of Balbiani body assembly. 2) We have identified Buc interacting proteins. We will study these interacting proteins, and conduct rescue based structure function analysis to identify Buc functional domains to understand the mechanism by which Buc regulates animal-vegetal polarity. 3) We will determine which factors mediate asymmetric buc mRNA localization and contribute to oocyte polarity. Understanding how the Balbiani body, a conserved oocyte asymmetric structure, forms in vertebrates will break new ground in the field of axis formation. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the mechanisms establishing these earliest oocyte asymmetries, which are conserved. In humans, mutations disrupting genes required to specify oocyte polarity or the first embryonic axis are expected to result in failed implantation or miscarriage due to severe developmental abnormalities. These most severe birth defects often are not detected in humans. In model systems such as zebrafish where fertilization and development of the embryo occur externally every egg that is produced can be examined for developmental abnormalities. Thus, this vertebrate genetic system allows access to maternally regulated developmental processes. An improved understanding of the essential maternal genes regulating early embryonic development in zebrafish will provide insight into the basis of birth defects and miscarriage, and facilitate comparison with human proteins. Studies of the Buc pathway are expected to be particularly relevant to abnormalities arising in very early pregnancy since buc mutant females produce eggs that are fertilized, but fail to specify the embryonic germ layers or axes. Completing these studies will provide insight into how Bucky ball regulates Balbiani body formation and oocyte asymmetry. These studies represent a first step toward deciphering the genes and mechanisms, mediating an evolutionarily conserved feature of primary oocyte development that is predicted to play fundamental roles in fertility, and in some vertebrates, establishment of the embryonic axes. PUBLIC HEALTH RELEVANCE: Prior to zygotic genome activation, vertebrate development depends on maternally supplied factors. However, the identity of the essential components and the molecular mechanisms underlying many maternally driven processes are not known. Mutations disrupting strict maternal-effect genes are viable. The mutant females are overtly normal, due to maternal function supplied by their mother. However, all of their progeny display the mutant phenotype regardless of their genotype. Although maternal products are essential for vertebrate development, only a small fraction of the vast numbers of vertebrate genes with maternal expression have been experimentally evaluated through genetic or by interference technologies. In each of these cases, insufficient maternal contribution results in early embryonic arrest, or profound developmental abnormalities. Similar genetic defects in humans would be expected to result in failed implantation or miscarriage before pregnancy is detected. Ten to twenty percent of known pregnancies result in miscarriage; however, when combined with undetected pregnancies, the actual percentage of pregnancies ending in miscarriage is estimated to be as high as 40-50% of all pregnancies, according to The March of Dimes, The American College of Obstetricians and Gynecologists, The Mayo clinic, and the National Institutes on Child Health and Development. Our research goal is to elucidate the genetic pathways and cell biological events that establish the first embryonic axis. We will use a combination of genetic, molecular, and cell biological approaches in the zebrafish model system. In humans, loss of function mutations in genes whose products are required to specify the first embryonic axis are expected to result in miscarriage due to severe developmental abnormalities. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the genetic basis of animal-vegetal axis formation, potentially illuminating the genetic basis of human birth defects, and early miscarriages of unknown etiology.